Intracellular transport is a fundamental biological process that is required for the proper distribution and positioning of organelles and macromolecular complexes in the cytoplasm of every eukaryotic cell. The goal of our project is to examine molecular mechanisms of intracellular transport using chemical genetics. This approach emulates classical genetics as a discovery tool, and involves the selection of chemical probes that can perturb a cellular function, identification of targets of these probes, followed by the use of the probes to clarify the contribution of known or novel proteins to the cellular function. We believe that the chemical genetic approach will be particularly effective in identifying and examining molecular mechanisms of vertebrate intracellular transport, where the application of genetic screens is difficult and approaches Perturbing protein function on time-scales much slower than the transport processes can be of limited use. We have identified a robust and efficient system that will allow the selection of compounds that either act on motor proteins, or proteins most directly involved in regulating motor protein function during intracellular transport. In the course of this work we will: (1) Use the Xenopus melanophore system to identify small molecule probes for intracellular transport. (2) Use secondary screens to select small molecules that either directly inhibit motor protein-driven organelle movement or target the regulatory mechanisms most closely associated with these movements. (3) Identify cellular targets, examine specificity and general applicability of chemical genetic probes for studying intracellular transport. The small molecules probes discovered and validated in our studies will be powerful tools to examine mechanisms of intracellular transport. It is likely that these small molecules will lead to the identification of new targets for therapeutic agents and improved treatments of human diseases.